European Journal of Immunology

Introduction: Tumor-reactive T cell receptors (TCRs) are promising candidates for the manufacturing of safe and effective adoptive TCR-T cell therapies, but their efficient identification remains a major challenge. We previously established an antigen-agnostic TCR-repertoire analysis workflow that prioritizes candidate TCRs based on intratumoral clonal expansion and high tumor-to-non-tumor (T/N) frequency ratios. Here, we investigated whether T/N-selected clonotypes show independent transcriptional and spatial features consistent with tumor association. Methods: Bulk and single-cell TCR repertoire sequencing of paired tumor and adjacent non-tumor tissues was used to identify clonotypes with high T/N ratios across 15 cancer cases, comprising six non-small cell lung cancers, five pancreatic cancers, two breast cancers, and two colorectal cancers. Candidate clonotypes were further characterized by paired single-cell TCR and gene expression profiling and, in two PDAC cases, by Xenium spatial transcriptomics to assess their cellular phenotype, spatial localization, and proximity to malignant cells. Results: High T/N ratios consistently marked T cell clonotypes with features of tumor reactivity across the studied solid tumor entities. The analysis included Cluster-A, a lead T cell cluster comprising highly similar TCRs shared among 14 HLA-A*02:01-positive patients in the present cohort. Cluster-A clonotypes were consistently enriched in tumor relative to matched non-tumor tissue, and previous functional studies demonstrated tumor recognition by Cluster-A members. In a focused analysis of a pancreatic cancer case, Cluster-A CD8 T cells were selectively detected in tumor tissue, preferentially localized near malignant epithelial cells, and displayed an antigen-experienced effector-memory phenotype with prominent expression of granzymes A and K (GZMA/GZMK). Importantly, in another PDAC case that lacked Cluster-A clonotypes, these features extended to independent clonotypes, revealing a continuous tumor-association axis in which increasing T/N ratios were associated with progressively closer localization to malignant cells and increased GZMA/GZMK expression. Thus, repertoire enrichment, spatial tumor proximity, and effector-associated transcriptional states converged across independent tumor-enriched clonotypes. Outlook: These findings support the T/N ratio as a robust primary criterion for identifying candidate tumor-reactive TCRs. Integration of single-cell and spatial transcriptomics provides supporting biological evidence of tumor association and a framework for prioritizing therapeutic TCR candidates when functional validation is limited.

Antiviral monoclonal antibodies (mAbs) rely on Fc-mediated effector functions, which depend on interactions with Fc receptors (FcRs) expressed on immune cells such as monocytes and dendritic cells (DCs). While combinations of antiviral mAbs with type I interferons (IFN-I) or IFN inducers are being explored to enhance therapeutic efficacy, the impact of these immunomodulators on FcR expression remains poorly understood. In this study, we investigated the effects of IFN-2, IFN-{beta}, and the IFN-inducer TAK-981 (a SUMOylation inhibitor) on FcR modulation and functional activation in human monocyte subsets and monocyte-derived DCs (moDCs). Using flow cytometry and imaging techniques, we mapped the expression of activating Fc{gamma}Rs (CD64, CD32A, CD16A) and neonatal FcR (FcRn) under steady-state and stimulated conditions. We found that IFN-I selectively downregulates CD16A expression in classical monocytes while preserving other FcRs, whereas TAK-981 reduces FcRn expression in classical monocytes without affecting CD16A. Additionally, IFN-{beta} and TAK-981 but not IFN-2, selectively enhanced Fc{gamma}RI expression in moDC. IFN-{beta} also induced a stronger maturation and functional activation program in monocytes and moDCs compared with IFN-2, whereas TAK-981 largely reproduced the IFN-{beta}-driven phenotype and secretory profile. Furthermore, we demonstrated that IFN-I signaling alters the spatial distribution of FcRs, with IFN-{beta} and TAK-981 promoting distinct redistribution patterns of Fc{gamma}Rs in monocytes and moDCs. These findings reveal a novel layer of FcR regulation, highlighting that IFN-I and IFN inducers differentially modulate FcR expression, localization, and functional activation in a cell-type- and stimulus-specific manner. Overall, this work provides a framework for understanding how IFN-I-based immunotherapies may influence Fc-mediated immune functions while preserving FcRn-dependent antibody persistence.

Advances in immunophenotyping of tumour-infiltrating lymphocytes (TILs) have improved our understanding of prognostic biomarkers and immune targets in human melanoma. However, whether the tumour-immune landscape in canine oral malignant melanoma (COMM) is concordant with that of human melanoma has not been properly defined. To address this gap, we developed a 16-colour spectral flow cytometry panel to characterise TIL phenotypic and functional profiles in COMM. Validation using mitogen-stimulated peripheral blood mononuclear cells from healthy dogs (n = 5) demonstrated robust identification of major T cell lineages, including regulatory T cells (Tregs) and memory subsets, and reliable evaluation of their activation and exhaustion status. COMM patients (n = 8) displayed a distinct protumour microenvironment, characterised by an increased proportion of Tregs, enrichment of tumour-specific exhausted-like T cells co-expressing programmed cell death protein 1 (PD-1) and tumour necrosis factor receptor 2 (TNFR2), and a reduction in cytotoxic CD8 and natural killer T (NKT) cell populations compared with tissue-resident (n = 4) and circulating (n = 8) lymphocytes. Analysis of additional solid tumours, including a mast cell tumour, nerve sheath tumour and adrenal cortical carcinoma, further supported the capability of the panel to identify similar patterns of immune dysregulation across diverse canine tumour landscapes. Collectively, this work describes the first detailed evaluation of canine TIL immunophenotyping using spectral flow cytometry and provides insights into the immunosuppressive mechanisms shaping the tumour microenvironment in COMM. These findings not only increase our understanding of canine tumour immunology but also identify potential immune targets and support ongoing comparative immuno-oncology efforts.

Interactions between intraepithelial lymphocytes (IELs) and the intestinal epithelium are central to mucosal homeostasis and disease. However, mechanistic in vitro studies describing their crosstalk in humans are limited by scarceness of primary material and insufficient knowledge about co-culture requirements. Here, we establish an autologous human duodenal IEL-organoid co-culture system encompassing expandable and bankable IEL and organoid protocols, with co-culture conditions that allow viability of both cell types. This system enables successive interrogation of lympho-epithelial interactions starting from minimal biopsy material. Under baseline conditions, CD45CD8CD103TCR{beta} IELs retain tissue-residency and effector features and induce an epithelial interferon response and chemokine production, without overt epithelial apoptosis. IL-15 and IL-21, essential cytokines involved in IEL-activation in intestinal enteropathies like celiac disease, increases granzyme B expression and interferon-{gamma} secretion but do not trigger epithelial cell death. However, enforcing IEL-epithelial contact using an anti-CD3-anti-Ep-CAM bispecific antibody induces epithelial apoptosis accompanied by increased tumor necrosis factor (TNF) and FAS-ligand (FASLG) secretion. These findings validate the platforms ability to resolve non-destructive and cytotoxic lympho-epithelial interaction and provide a tractable system for studying intestinal inflammation and immune-mediated epithelial cell death.

Background Vitiligo is an acquired depigmenting disorder characterised by melanocyte loss. While immune responses against melanocytes are implicated in its pathogenesis, melanocyte detachment, melanocyte senescence and aberrant epidermal differentiation are increasingly characterised as additional pathological features. Granzyme B (GzmB), classically recognised as an intracellular effector of perforin-dependent cytotoxicity, is increased in vitiligo lesions; however, its extracellular contribution to vitiligo pathology remains unclear. Objectives To determine whether extracellular GzmB contributes to depigmentation and epidermal pathological features relevant to vitiligo, and to define its cellular source and underlying mechanisms. Methods Human vitiligo skin samples, murine depigmentation models and cultured human epidermal cells were analysed. The functional effects of extracellular GzmB were examined by subcutaneous administration of recombinant GzmB in mice. A rhododendrol (RD)-induced leukoderma model was used to evaluate the disease relevance of GzmB and the therapeutic effect of topical GzmB inhibition with VTI-1002. Results GzmB-positive cells were markedly increased in active vitiligo lesions and were predominantly associated with tryptase-positive cells, with limited co-distribution with CD8 or perforin. Vitiligo lesions also showed melanocyte detachment, increased p16INK4A-positive melanocytes and aberrant keratinocyte differentiation. These pathological features were recapitulated in vivo by extracellular GzmB administration, accompanied by focal depigmentation. In cultured melanocytes, GzmB exerted no cytotoxic effects but reduced attachment strength and induced a senescence-associated phenotype characterised by decreased extracellular matrix- and adhesion-related gene expression, increased p16INK4A level, elevated senescence-associated {beta}-galactosidase activity and activation of transforming growth factor-{beta}/SMAD signalling. In keratinocytes, extracellular GzmB promoted aberrant differentiation associated with activation of p53 signalling. Topical inhibition of GzmB in RD-induced leukoderma attenuated depigmentation progression, melanocyte detachment, melanocyte senescence and abnormal epidermal differentiation. Conclusions Extracellular GzmB promotes depigmentation associated with inducing melanocyte detachment, melanocyte senescence and aberrant keratinocyte differentiation. These findings identify extracellular GzmB as a previously underrecognised pathogenic mediator and potential therapeutic target in vitiligo-associated depigmentation.

The non-canonical NF-kappa B pathway, in which NFKB2 plays a central role, is critical for lymphoid organ development and immune homeostasis. Pathogenic NFKB2 variants have been strongly associated with a broad spectrum of human pathologies, notably immunodysregulation syndromes, characterized by recurrent infections, autoantibody production and endocrine abnormalities. The mechanisms underlying the resulting immune defects remain incompletely understood. In the present study, we characterized the immune phenotype of a novel rat model harboring a Nfkb2 non-sense mutation located in the c-terminal region of the gene (c.2602T>A; p.Tyr867*, Nfkb2p.Tyr867*). This variant has been identified in patients with a familial form of common variable immunodeficiency (CVID) presenting with autoimmunity manifestations. Using this model, we demonstrate that mutated NFKB2 resulted in profound alterations of the thymic microenvironment, including reduced K5+ medullary thymic epithelial cells and decreased AIRE expression, accompanied by altered thymocyte development and impaired thymic regulatory T populations. Peripheral lymphoid organization was also profoundly affected, with absence of lymph nodes, defects in the splenic marginal zone, and alterations of CD4+ and CD8+ T-cell homeostasis, including impaired Treg populations and reduced ICOS expression on CD4+ T cells. The B-cell compartment was markedly altered, with impaired B-cell maturation, characterized by accumulation of IgM+IgD- transitional cells and a marked reduction of IgD+ mature B cells. These abnormalities were associated with impaired humoral immunity and complete absence of circulating immunoglobulins. Collectively, our findings define a systemic combined immunodeficiency phenotype in the Nfkb2-mutated rat, highlighting its utility for studying the role of non-canonical NF-kB signaling in immune system organization and adaptative immunity.

Unconventional T cells recognize non-peptide antigens presented by molecules other than the major histocompatibility complex (MHC) proteins. Among unconventional T cells, natural killer T (NKT) cells, which recognize CD1d-lipid complexes, mucosal-associated invariant T (MAIT) cells, which recognize MR1-metabolite complexes, and {gamma}{delta} T cells, are thoroughly studied. CD1b presents self- and mycobacterial lipids to relatively understudied T cell subsets. Like MAIT cells and type I NKT cells, CD1b-specific cells include subpopulations with conserved TCRs. Consequent to their recognition of a nearly monomorphic antigen-presenting molecule, CD1b-specific T cells might share innate-like features with MAIT, type I NKT, and {gamma}{delta} T cells. Due to their low frequency in the peripheral blood, CD1a-, CD1b-, and CD1c-specific T cells have been studied predominantly as in vitro-expanded clones, so even basic information about their native ex vivo immunophenotypes is lacking. Here, we sort and transcriptionally profile ex vivo two T cell populations that recognize CD1b presenting mycobacterial mycolipids and compare them with conventional CD4 and CD8 T cells, {gamma}{delta} T cells, NK cells, MAIT cells, and NKT cells. We show that both the invariant TCR-expressing CD4+, CD1b-GMM-specific germline encoded mycolyl-reactive (GEM) T cells, as well as the diverse TCR-expressing CD1b-GMM-specific T cells, are transcriptionally closer to conventional T cells than to the innate-like T cell populations {gamma}{delta}, MAIT and type I NKT cells. Thus, despite their recognition of non-polymorphic antigen presenting molecules, CD1b-specific T cells show adaptive rather than innate-like transcriptional features.

Maintaining brain homeostasis is crucial for proper function of the central nervous system and has traditionally been attributed to neuronal and glial interactions. However, recent research highlights the essential role of brain-resident immune cells in this process. Our study characterizes brain-specific CD8+ T cells and elucidates their significant contribution to brain homeostasis and behavior. We identified a distinct population of CD8+ T cells that infiltrates the brain during early development, undergoes clonal expansion, and acquires effector memory-like characteristics through interactions with microglia. Notably, the absence of these cells results in hyperactivation of neuronal activity and abnormal behaviors, due to loss of regulation of interferon-gamma (IFN-{gamma}) secreted by CD8+ T cells. Our findings demonstrate that IFN-{gamma} secreting brain-specific CD8+ T cells are crucial for maintaining the physiological level of neuronal excitability and normal behavioral patterns. This study provides novel insights into neuroimmune interactions, emphasizing the critical role of CD8+ T cells in sustaining brain function and behavior.

BackgroundStrategies to define human monocytes by flow cytometry vary considerably across studies. Recently, toll-like receptor 2 (TLR2) has been proposed as a marker to identify "all monocytes" in human peripheral blood. However, the TLR2-defined monocytes also contained a previously ignored TLR2posCD14dim/negCD16neg population, which we termed the unclassified subset (UCS). MethodsPeripheral blood mononuclear cells (PBMCs) from healthy donors and patients with multiple myeloma (MM) or monoclonal gammopathy of undetermined significance (MGUS) were analyzed by multiparameter flow cytometry using TLR2pos gating. PBMCs from additional healthy donors were analyzed to characterize the UCS population, including the impact of using either TLR2pos or a negative selection-based gating strategy. ResultsThe TLR2pos CD14dim/neg CD16neg UCS population was present in healthy controls, MGUS, and MM patients. The UCS expressed the monocyte-macrophage scavenger receptor CD163 and was significantly reduced in MM patients compared to healthy donors (P<0.002). Further phenotypic characterization in healthy blood donors revealed that approximately 80% of UCS cells expressed CD163 at levels comparable to classical monocytes, yet phenotypically resembled CD163pos dendritic cells (DCs). Importantly, gating strategies influenced the composition of the UCS: negative selection-based gating captured all DC subsets, whereas TLR2pos gating primarily included CD1cpos DCs that were highly CD163pos. ConclusionsThese findings demonstrate that circulating CD163pos CD1cpos DCs are included in the TLR2pos cell population previously described as exclusively monocytes, highlighting the impact of gating strategy on monocyte subset identification. Further, the lower level of TLR2pos CD14dim/neg CD16neg CD163pos cells in MM patients may represent decreased levels of circulating DCs that may contribute to the immune dysregulation in this disease.

The transcriptional and cell repertoire changes of lymph node (LN) T cells following influenza vaccination have been under-investigated across different ethnicities. Draining (dLN) and non-draining (ndLN) axillary LN cells from 17 participants with genotypically varied African or Asian ancestry (ISRCTN13657999) were collected by fine needle aspiration (FNA) before, 5 days and 42 days after adjuvanted influenza vaccination and analysed by single-cell RNA-sequencing (scRNA-seq). The early dLN transcriptional profile and cellular dynamics were distinct from the ndLN; BACH2 expressing T cells were highly induced, exhibiting stem-like and unique quiescence signatures. Proliferating T follicular helper cells (Tfh) were enriched in dLN and Th1-like Tfh predominated with highly induced transcriptional changes. The BACH2+ T cell state and Th1-like Tfh cells, transcriptionally distinct from germinal centre (GC) T cells, were co-localised with LAMP3+ dendritic cells in the T cell zone of publicly available LN datasets, a region that was also distinct from GC Tfh niches. Together, these findings indicate that the induction of a robust, proliferative, and transcriptionally diverse Tfh response, alongside a discrete subset of BACH2+ T cells, represents a marked feature of the early T cell zone immune response to adjuvanted influenza vaccine in the dLN.

The skull bone marrow axis has recently emerged as an important source of immune cells during neuroinflammation, including in the experimental autoimmune encephalomyelitis (EAE) , the mouse model of multiple sclerosis. The cribriform plate bone marrow (cpBM), is a portion of the ethmoid bone of the skull of mice, and is positioned next to nasal regions. At the cribriform plate olfactory nerves pass through the skull and recent evidence supports that it represents an important immune niche with links to CSF outflow and connections to cpBM. Previous data supports cellular composition of the ethmoid bone marrow in mice is altered in neuroinflammation suggesting it senses inflammatory cues, but the exact nature of these changes is still unclear. Here using scRNAseq and flow cytometry of the ethmoid bone marrow region we test how the region responds to EAE. In addition we also compare the cpBM region with systemic bone marrow in the femur to understand region specific differences. We report that neuroinflammation creates a regulatory response at these sites and implicates the cpBM in controlling these effects. A better understanding of the cribriform niche may offer new treatments for neuroinflammatory disease

CD11c+CD21- B cells are overrepresented in rheumatoid arthritis (RA). Here we delineate their functional state at site of inflammation and its molecular basis. Phenotypic profiling of paired blood and synovial fluid from patients with anti-citrullinated protein antibody (ACPA) seropositive RA (N=12), seronegative RA (N=10) and spondyloarthritis (N=8), demonstrated a striking enrichment of CD11c+CD21- B cells in synovial fluid in all three diseases (average of CD19+ cells: 67%, 49% and 51%). In ACPA-positive RA we identified increased proportions of CD11c+IgG+CD27low cells and several stages of pre-plasmablasts. The CD11c+CD21- cells had a phenotype indicating BCR activation, proliferation, and antigen presentation. This B-cell profile was mirrored by a LAG3+CTLA4+TIGIT+ peripheral helper T-cell program in ACPA-positive RA, contrasting a CCR6+ Th17-skewing in seronegative RA. Single-cell spatial proteomics revealed a unique CD11chighCD21 low B-cell surface architecture including a BCR co-receptor-tetraspanin signature with CD72-CD81 co-localization, which was elevated in RA. CD11c+ cells also displayed CD44-CD52/CD59 and MHCII-CD40/CD84 immune-modules absent in other B-cell subsets. In summary, CD11c+CD21- B cells are highly diverse, and while an activated antigen-experienced synovial profile was shared between disease groups, ACPA+ RA showed evidence of increased extrafollicular plasmablast differentiation. Surface receptor organization implicated a favoured BCR responsiveness, complement resistance and antigen presentation.

Background: Chimeric Antigen Receptor (CAR) T-cell therapy has transformed the treatment of hematologic malignancies, yet, its broader clinical application often faces major challenges, including slow expansion rates, variable transduction efficiency, exhaustion, and functional heterogeneity. Recent studies have demonstrated that a Synthetic Immune Niche (SIN) composed of immobilized CCL21 and ICAM1 promotes the proliferation of murine and human T-cells while preserving their cytotoxic potency. In this study, we explored the capacity of immobilized CCL21 and ICAM1 to enhance the production of highly potent CAR T-cells by facilitating both their expansion and cytotoxic capacity. Methods: CD19-directed CAR T-cells were generated from PBMCs of healthy donors using a clinical-grade protocol. Following retroviral transduction, the cells were expanded on CCL21 and ICAM1 coated plates, or on uncoated control plates. The effects of the SIN treatment on CAR T-cell expansion, morphology, physical properties, phenotypic markers, and potency were systematically evaluated. Results: SIN exposure significantly enhanced CAR T-cell expansion, achieving a 9.3-fold higher total cell yield by day 13, compared with control cultures. This proliferative advantage persisted even after withdrawal of the cells from the synthetic niche on day 10. SIN stimulation preferentially expanded the CAR-transduced population, increasing CAR T-cell frequencies from 45.4% to 77.6%, resulting in a 14.6-fold increase in the absolute number of CAR T-cells compared with untreated cultures. Morphological and phenotypic analyses revealed distinct activated cell morphology, manifested by increased cell size, polarity and granularity, and elevated expression of the activation markers CD137 and CD69. Importantly, CAR T-cells transiently exposed to the SIN retained cytokine secretion and cytotoxic activity comparable to that of continuously SIN-treated cells, indicating that the SIN effect is persistent. Overall, SIN-conditioned CAR T-cells displayed robust antigen-dependent IFN-{gamma} secretion and cytotoxicity against CD19-expressing target cells. Conclusion: Stimulation of CAR T-cells with a synthetic immune niche consisting of immobilized CCL21 and ICAM1 enhances overall expansion while selectively enriching the CAR-transduced population, thereby substantially increasing both the number and prominence of therapeutically relevant CAR T-cells. This effect is accompanied by a persistent activation phenotype and high functional potency. We propose that incorporating SIN stimulation into CAR T-cell manufacturing represents a simple and scalable strategy for improving CAR T-cell yield while maintaining high cytotoxic efficacy.

Group 2 innate lymphoid cells (ILC2s) regulate type 2 immune responses partly by recruiting eosinophils, but the mechanisms underlying this process remain incompletely understood. Although both ILC2s and type 2 T helper (Th2) cells are capable of expressing IL-5, a cytokine critical for recruiting eosinophils, ILC2s are more potent than Th2 cells in this process. Here, we show that the transcription factor Bhlhe40 promotes GM-CSF production in ILC2s, and Bhlhe40 expression in ILC2s is required for efficient eosinophil recruitment during both papain- and helminth-induced type 2 immune responses. However, Bhlhe40 deficiency in ILC2s had no effect on the production of classical type 2 cytokines, including IL-4, IL-5 and IL-13, despite Bhlhe40 being required for type 2 cytokine production by Th2 cells. Furthermore, in contrast to ILC2s, Th2 cells produced little GM-CSF and administration of GM-CSF rescued eosinophil recruitment in ILC2-specific Bhlhe40-deficient mice. In addition to promoting GM-CSF expression, Bhlhe40 repressed IL-10 production in ILC2s as it did in Th2 cells, particularly during chronic inflammation. Single-cell transcriptomic analyses further supported this regulatory network, and the ChIP-Seq data revealed direct binding of Bhlhe40 to the enhancer and promoter regions within the Il10 and Csf2 loci. Collectively, our findings show that Bhlhe40 exerts distinct gene regulatory functions in ILC2s and Th2 cells, and Bhlhe40 modulates the inflammatory and anti-inflammatory functions of ILC2s by promoting GM-CSF while suppressing IL-10 production.

Fragment crystallizable gamma receptor (Fc{gamma}R)-induced signaling is a crucial process that determines the cellular response to immune complexes (IC) in autoimmune diseases. In several diseases including pemphigoid diseases (PD), such as epidermolysis bullosa acquisita (EBA), or rheumatoid arthritis (RA), neutrophils are prominently involved as effector cells, while others such as immune thrombocytopenia (ITP) are independent of neutrophils. At the same time, most diseases are commonly treated by broad-range immunosuppression accompanied by severe risk for adverse effects. Signal transduction inhibitors (STIs) have been successfully applied in cancer therapy. However, their use in autoimmune diseases is an emerging, but so far understudied potential treatment avenue. Therefore, we screened a target-selective compound library consisting of 155 STIs in a neutrophil-based assay and conducted a multiplex kinase activity profiling with IC-stimulated neutrophils. Thus, we found novel potential therapeutic targets that were validated both in vitro in functional neutrophil assays and in vivo in murine models of EBA Here, we demonstrate that both systemic and topical treatment with several individual STIs is effective in a prophylactic approach in these models. Furthermore, therapeutic treatment with the BTK inhibitor ibrutinib in the immunization-induced EBA model reduced disease severity by approximately 85 % and showed efficacy in additional experimental models of EBA, arthritis, and ITP. Together, the present study contributes to the elucidation of Fc{gamma}R-dependent signaling in neutrophils and identifies multiple novel promising treatment options including inhibition of PLC, PDK-1, PKC, p38, DNA-PK, KSP, c-Met, TBK-1 and BTK for IC-mediated autoimmune diseases.

Production of aberrant glycans by cancer cells constitutes a key immunosuppressive strategy to avoid destruction by immune cells. Although sialic acid-containing glycans are known to dampen the activation of lymphocytes, including Natural Killer (NK) cells, the role of fucose-containing glycans remains poorly characterized. In this work, we explored the role of Lewis X (LeX) in cancer cell-NK cell interactions. We induced ectopic expression of FUT9, an 1-3/4-fucosyltransferase, in two colorectal cancer cell lines and showed this enzyme only synthesized LeX structures but not sialyl-LeX. FUT9 introduction was not associated with altered MHC class I surface expression, nor with CD2 (which has been proposed as a receptor for LeX) binding to cancer cells. By inhibiting fucosylation we could demonstrate that CD2 binding was furthermore independent of surface fucosylated glycans in three independent cell lines. Lastly, FUT9/LeX had a limited role in cancer cell destruction and expression of activation markers by NK cells. Overall, our study suggests that, unlike sialylated glycans, 1-3/4-fucosylated glycans have limited impact on cancer cell evasion of NK cell-mediated destruction.

Osteocytes are the most abundant cells in bone and are increasingly recognised not only for their role in skeletal remodelling and inflammatory signalling but also for their potential involvement in immune responses. In this study, we searched available gene expression datasets of human primary osteocyte-like cells exposed acutely to Staphylococcus aureus and identified significantly induced expression of key genes related to antigen processing and presentation. We then confirmed that human bone explant-derived osteoblastic cells, representative of a mature osteoblast-pre-osteocyte stage, expressed, as expected, high cell surface levels of major histocompatibility complex (MHC) Class I but also, low basal levels of the MHC Class II family member, HLA-DR. However, confocal imaging revealed high expression of MHC Class II molecules and the peptide-loading chaperone HLA-DM within the lysosomal compartments, consistent with canonical antigen-processing machinery. Differentiation towards a mature osteocyte phenotype increased MHC Class II protein levels and maintained expression of intracellular HLA-DM. Exposure of mature osteocyte-like cells to S. aureus further up-regulated both intracellular and cell surface MHC Class II expression. Demonstrative of antigen presenting cell functionality, S. aureus-exposed osteocytes induced autologous CD4+ T cell proliferation. Furthermore, MHC Class II expression in osteocytes was detected in bone sampled from patients with periprosthetic joint infections, providing evidence that these mechanisms operate in vivo. Together, our findings reveal that human osteocytes are capable of inducible MHC Class II-associated antigen presentation in response to bacterial challenge, pointing to a novel role for osteocytes in adaptive immune surveillance within bone.

Dendritic cells (DCs) are key orchestrators of anti-tumor adaptive immune responses. Their function is tightly regulated by galectins, a family of carbohydrate-biding proteins that decode extracellular glycans into intracellular signaling. Here, we show that exposure to melanoma-conditioned media (CM) induces loss of galectin-9 (gal-9) at the DC surface. Notably, gal-9 loss, independent of transcriptional regulation, was associated with the acquisition of an immunosuppressive phenotype in CD14cDC2 cells following tumor exposure, with higher-molecular weight fractions of the melanoma secretome as mediators. Notably, gal-9 depletion was mirrored with a reduction in gal-9 ligands on the cell surface, particularly GalNAc-containing glycoepitopes, pointing towards a melanoma-exploited gal-9-glycan axis as a DC-evasive strategy. Interestingly, restoring gal-9 surface levels in CM-exposed CD14cDC2 cells prevented the expansion of regulatory T cells (Tregs), postulating gal-9 as a novel immunomodulatory molecule in DC-mediated Treg induction during melanoma progression. Altogether, our data suggest that melanoma-derived factors remodel the DC glycan-gal-9 axis to enhance T cell differentiation towards regulatory phenotypes and dampen anti-tumor immunity. This identifies the gal-9/glycan axis in CD14cDC2 cells as a vulnerable node in melanoma immune evasion and as a potential therapeutic target.

During chronic infection, the persistence of antigen and inflammation leads to the differentiation of CD8+ T cells into an exhausted state characterised by expression of inhibitory receptors (IRs) and the progressive loss of T cell functions. Among the different subsets of exhausted CD8+ T (Tex) cells, Tex progenitors expressing SLAMF6 and the transcription factor TCF-1 (TCF-1+) give rise to Tex effector-like cells expressing CX3CR1 and Tex terminal cells expressing CD101. PD-1/PD-L1 blockade acts on TCF-1+ Tex progenitor cells and promotes their differentiation into Tex effector-like cells. The molecular events controlling CD8+ Tex cell differentiation are still poorly defined. As Notch signaling may be sustained during chronic infection by persistent TCR stimulation and inflammation, we tested whether Notch signaling influences CD8+ T cell exhaustion. Using mice lacking (N1N2{triangleup}/{triangleup}) or not (N1N2fl/fl) Notch1/2 expression only in mature CD8+ T cells, we showed that the absence of Notch signal causes severe CD8+ T cell exhaustion during chronic LCMV infection. N1N2{triangleup}/{triangleup} Tex cells express higher levels of IRs and are less functional when compared to their wild-typee counterpart. In the absence of N1N2 receptors, Tex progenitor and Tex terminal cells accumulate and Tex cells cannot be reinvigorated by PD-1/PD-L1 blockade. We further demonstrated that Notch signaling is essential to promote the differentiation of Tex progenitors into Tex effector-like cells. Moreover, Notch signals, provided by stromal cells expressing the ligands Delta-like 1 and 4, are necessary during all stages of the infection to prevent severe exhaustion. Single-nucleus RNA and ATAC multiome profiling identifies Notch signaling as a critical role on effector transcriptional programming in exhausted CD8 T cells. Loss of Notch signaling impairs transcriptional program associated with migration and perception of CD4+ T cell help. Together, these alterations drive the differentiation of Tex progenitor cells toward a terminally exhausted Tex fate.

APECED (Autoimmune PolyEndocrinopathy Candidiasis Ectodermal Dystrophy) is a rare syndrome of multi-organ autoimmunity driven by the presence of self-reactive T cells and autoantibodies caused by mutations in the gene Autoimmune regulator (AIRE). Compared to the well-defined role of AIRE in establishing and maintaining T cell central tolerance, less is known about how AIRE deficiency impacts B cell phenotypes that may contribute to a breakdown in peripheral B cell tolerance. Here we analyzed serum and peripheral blood cells from APECED patients and healthy donors using autoantibody profiling, proteomics, flow cytometry, scRNAseq based subset analysis, BCRseq, and autoantigen binding assays finding significant changes to the APECED B cell compartment. We show that while Naive and Transitional B cells are reduced, alternatively activated B cell subsets are expanded in APECED patients including IgM CD27+ and class switched Atypical B cells which exhibit BCR chain features prone to autoreactivity. Antibodies derived from either APECED or Healthy class switched atypical B cells bind autoantigens at significantly higher rates than control IgG B cells. Serum autoantibodies and proteomics highlight APECED common and patient variable changes. These results together show that APECED causes a compositional shift towards subsets that may promote broken B cell tolerance and autoimmunity.